Cecilia Bartolucci

Three-dimensional structure of a complex of galanthamine (Nivalin) with acetylcholinesterase from Torpedo californica: implications for the design of new anti-Alzheimer drugs

The 3D structure of a complex of the anti‐Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. The X‐ray structure, at 2.5Å resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein–ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl‐binding pocket and the principal quaternary ammonium‐binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84. A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest‐energy structure has a root mean square deviation of 0.5Å with respect to the corresponding crystal structure of the complex. The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure‐based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182–191.

Accurate prediction of the bound conformation of galanthamine in the active site of Torpedo californica acetylcholinesterase using molecular docking

The alkaloid (-)-galanthamine is known to produce significant improvement of cognitive performances in patients with the Alzheimers disease. Its mechanism of action involves competitive and reversible inhibition of acetylcholinesterase (AChE). Herein, we correctly predict the orientation and conformation of the galanthamine molecule in the active site of AChE from Torpedo californica (TcAChE) using a combination of rigid docking and flexible geometry optimization with a molecular mechanics force field. The quality of the predicted model is remarkable, as indicated by the value of the RMS deviation of approximately 0.5A when compared with the crystal structure of the TcAChE-galanthamine complex. A molecular model of the complex between TcAChE and a galanthamine derivative, SPH1107, with a long chain substituent on the nitrogen has been generated as well. The side chain of this ligand is predicted to extend along the enzyme active site gorge from the anionic subsite, at the bottom, to the peripheral anionic site, at the top. The docking procedure described in this paper can be applied to produce models of ligand-receptor complexes for AChE and other macromolecular targets of drug design.

Probing Torpedo californica acetylcholinesterase catalytic gorge with two novel bis-functional galanthamine derivatives.

N-Piperidinopropyl-galanthamine (2) and N-saccharinohexyl-galanthamine (3) were used to investigate interaction sites along the active site gorge of Torpedo californica actylcholinesterase (TcAChE). The crystal structure of TcAChE-2 solved at 2.3 A showed that the N-piperidinopropyl group in 2 is not stretched along the gorge but is folded over the galanthamine moiety. This result was unexpected because the three carbon alkyl chain is just long enough for the bulky piperidine group to be placed above the bottleneck (Tyr121, Phe330) midway down the gorge. The crystal structure of TcAChE-3 at 2.2 A confirmed that a dual interaction with the sites at the bottom, and at the entrance of the gorge, enhances inhibitory activity: a chain of six carbon atoms has, in this class of derivatives, the correct length for optimal interactions with the peripheral anionic site (PAS).

Inhibition of human leukocyte elastase by chemically and naturally oversulfated galactosaminoglycans

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

Kinetics of Torpedo californica acetylcholinesterase inhibition by bisnorcymserine and crystal structure of the complex with its leaving group.

Natural and synthetic carbamates act as pseudo-irreversible inhibitors of AChE (acetylcholinesterase) as well as BChE (butyrylcholinesterase), two enzymes involved in neuronal function as well as in the development and progression of AD (Alzheimers disease). The AChE mode of action is characterized by a rapid carbamoylation of the active-site Ser(200) with release of a leaving group followed by a slow regeneration of enzyme action due to subsequent decarbamoylation. The experimental AD therapeutic bisnorcymserine, a synthetic carbamate, shows an interesting activity and selectivity for BChE, and its clinical development is currently being pursued. We undertook detailed kinetic studies on the activity of the carbamate bisnorcymserine with Tc (Torpedo californica) AChE and, on the basis of the results, crystallized the complex between TcAChE and bisnorcymserine. The X-ray crystal structure showed only the leaving group, bisnoreseroline, trapped at the bottom of the aromatic enzyme gorge. Specifically, bisnoreseroline interacts in a non-covalent way with Ser(200) and His(440), disrupting the existing interactions within the catalytic triad, and it stacks with Trp(84) at the bottom of the gorge, giving rise to an unprecedented hydrogen-bonding contact. These interactions point to a dominant reversible inhibition mechanism attributable to the leaving group, bisnoreseroline, as revealed by kinetic analysis.

Isolation and Quantitation of Hyaluronan Tetra- and Hexasaccharide by Anion Exchange HPLC

Abstract A high performance liquid chromatographic method for the isolation and quantitation of the the tetra- and hexasaccharide derived from enzymatic depolymerization of hyaluronan by bovine testicular hyaluronidase (EC 3. 2. 135) is described in this paper. This method is well suited for the HPLC analysis of depolymerization reactions of hyaluronan, either alone or in competition with chondroitin sulphate, another glycosaminoglycan which is also a substrate for the hyaluronidase. Using a polymeric-based DEAE column with 50 mM phosphate buffer (pH 3.5) as mobile phase, complete separation of hyaluronan tetra- and hexasaccharide was achieved both in analytical and preparative scale. Under these conditions oligomers higher than hyaluronan-hexasaccharide and the chondroitin sulphate depolymerization products were strongly retained and were not eluted. Their complete elution was obtained in a single desorption step with 1.0 M potassium chloride in the eluent. Changes of the ionic strength as well as of the...